The present invention relates to assaying compounds containing primary and secondary amino groups and more particularly to novel compounds which react with primary or secondary amines to form adducts amenable to fluorometric detection techniques.
The biological role of peptides, either of endogenous or exogenous origin, is of great interest to researchers. This interest has been particularly evident ever since the discovery of the body's natural opioid peptides including .beta.-endorphin, Met-enkephalin and Leu-enkephalin. Interest has also been enhanced by the discovery that endogenous peptides take part in many varied biological processes, including regulation of blood pressure and neurotransmission, which is regulated by peptides such as atrial natriuretic factor and neuropeptide. Such discoveries, coupled with recent advances in biotechnology, have led to the active investigation by many pharmaceutical companies of the possibility of developing drugs based on naturally occurring peptides or their synthetic analogs.
Of course, the study of the biological role of various peptides for possible future pharmaceutical applications requires techniques enabling isolation and precise quantification of such peptides in biological fluids and tissues of interest. Isolation, or selectivity, involves utilization of a technique for separating a given peptide from other peptides as well as from other biological compounds. In recent years, the technique of high performance liquid chromatography (HPLC) has emerged as the dominant technique available for fractionating complex biological samples and providing the required selectivity. Quantification involves determining the amount of the isolated peptide present in a biological sample.
Unfortunately, amino acids and peptides present in biological fluids do not possess the physical properties necessary to allow their quantification at their naturally occurring concentration levels, i.e., at concentrations of 10.sup.-9 mole/liter (nanomoles/liter) or less. One frequently employed approach to overcome this problem is to chemically modify (derivatize) the peptide or amino acid, such as by fluorescent labelling, prior to or after isolating the various peptides or amino acids by HPLC. In peptide and amino acid analysis, the chemical handle most amenable to chemical derivatization is the primary or secondary amino group of the N-terminus for a peptide or the .alpha.-amino group for any amino acid.
Several properties should be possessed by a derivatization agent used to fluorescently label peptides or amino acids. First, the derivatization reagent should be capable of reacting with both primary and secondary amines. Secondly, the reagent should be fluorogenic, i.e., not itself fluorescent but capable of forming a fluorescent adduct upon reaction with the peptide or amino acid. Thirdly, the reagent should be capable of forming a chemically stable adduct with a primary or secondary amine. Fourthly, the adduct formed should exhibit a high fluorescence efficiency in the aqueous/organic solvent systems utilized in HPLC. Finally, the reagent should react with the primary or secondary amine compound under relatively mild reaction conditions.
Over the years, a number of fluorogenic derivatization reagents for amines have been developed. For example, ortho-phthalaldehyde (OPA) and fluorescamine have been employed for the fluorogenic derivatization of primary amines. However, such compounds do not form fluorescent derivatives with secondary amines. Additionally, OPA only forms fluorescent derivatives with amino acids and not peptides whereas fluorescamine forms two products with amino acids. Other fluorogenic derivatization reagents for amines include dansyl-chloride (Dans-Cl) and 4-chloro(fluoro)-7-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl(F)]. However, Dans-Cl usually does not react to give quantitative yields. Additionally, excess Dans-Cl forms a hydrolysis product, Dans-OH, which has the potential for interfering with the dansylated amines. NBD-Cl and NBD-F amine derivatives exhibit substantially compromised fluorescence quantum efficiencies in media containing a large fraction of water which, unfortunately, is commonly encountered in HPLC.